Recombinant human cytomegalovirus protease with a C-terminal (His)6 extension: purification, autocatalytic release of the mature enzyme, and biochemical characterization.

Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics. To obtain large amounts of native protease, a 268-amino-acid polypeptide with a hexahistidinyl tag at the C terminus was expressed in Escherichia coli. The first 262 amino acids of the recombinant protein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteolysis at that site. The hexahistidinyl tag was placed downstream of amino acid 262 of the native CMV PR sequence. In this design, the Ala-Ser bond at amino acids 256-257 constitutes a site naturally cleaved by the protease during capsid maturation. The 268-amino-acid polypeptide with the (His)6 tag was expressed at high levels in E. coli as inclusion bodies. After solubilization of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni2+ affinity chromatography. The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage of the Ala-Ser bond at amino acids 256-257 to remove 12 amino acids including the (His)6 tag from the C terminus of the protein. This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C terminus. Approximately 40 mg of purified CMV PR was obtained per liter of cell culture using this strategy. The enzymatic activity of CMV PR purified from inclusion bodies and refolded to an active enzyme was similar to the enzymatic activity of CMV PR expressed as a soluble protein in E. coli. In addition, the refolded CMV PR could be crystallized for X-ray diffraction.

Does fortune favour the bald? Psychological correlates of hair loss in males.

In order to investigate the relationship between male hair loss and psychological distress, 182 men were recruited who had a wide range of ages and hair loss varying from none to severe. Care was taken to ensure that hair loss and age were uncorrelated in the sample. Multiple regression was used to predict possible consequences of baldness, controlling for age, and examining the interaction between baldness and age to see if consequences were especially severe in cases of premature baldness. Increasing degrees of hair loss were associated with loss of self-esteem, depression, introversion, neuroticism and feeling unattractive. These effects were more marked for young men in the case of self-esteem, introversion and feeling unattractive.

Immunisation against HSV-1 keratitis with a synthetic gD peptide.

The authors tested the protective efficacy of, and the immune response to, immunisation with a synthetic peptide of glycoprotein D (gD) of HSV-1 in a murine model of herpes stromal keratitis (HSK). HSV-1 susceptible A/J mice were immunised subcutaneously with a peptide corresponding to the N-terminal epitope gD(5-23) prior to corneal HSV-1 challenge. Divergent immunisation protocols were compared for their protective potency, their ability to prevent the establishment of latency in the trigeminal ganglion, and their effect on the immune system. Low dosages (31 micrograms) of gD(5-23) protected against encephalitis and HSK. Protective efficacy was higher when gD(5-23) was coupled to the carrier protein keyhole limpet haemocyanin (KLH) and was emulsified with adjuvant. Latent infection was found in all control mice but in only 50-75% of immunised mice. The most potent protection was correlated with anti-HSV-1 neutralising antibodies of IgG1 and IgG2a isotypes, but free gD(5-23) protected in the absence of anti-HSV-1 antibodies. Our results suggest that immunisation with gD(5-23) stimulates both humoral and cellular immune mechanisms which protect against HSV-1 keratitis.

[T-cell-induced prevention of HSV-1 keratitis by immunization with the synthetic peptide of glycoprotein D]. / T-Zell-vermittelter Schutz gegen HSV-1-Keratitis durch Immunisierung mit synthetischem Peptid des Glykoprotein D.

C.AL-20 mice susceptible to herpes simplex virus (HSV) were protected against HSV keratitis (HSK) and encephalitis by subcutaneous immunization with synthetic peptide corresponding to the N-terminal amino acid residues 5-23 of HSV glycoprotein D, which is a dominant immunogen of HSV-1. Protection against HSV was related to a potent humoral anti-HSV response. FACScan analysis revealed that CD4+V beta 8(1.2)+ cells in the spleen were markedly decreased 2 days after HSV challenge, and CD8+ cells were increased. Numerous CD4+ and V beta 8(1.2)+ cells were found in the corneal tissue from HSV-infected sham-immunized mice; no such cells were seen in gD(5-23) immunized mice. No cytotoxic cells were detected in the corneas or spleens of gD(5-23) immunized mice, and these mice had decreased DTH responses. Protection against HSV through immunization with gD(5-23) involves humoral and cellular immune mechanisms. CD4+V beta 8(1.2)+ maybe critical in mediating the pathology of HSK. CD8+ cells may be protective by non-cytotoxic mechanisms. Our results suggest that gD peptides may be potent as vaccines against HSV.

Purification of a recombinant human respiratory syncytial virus chimeric glycoprotein using reversed-phase chromatography and protein refolding in guanidine hydrochloride.

FG glycoprotein is a recombinant chimeric protein consisting of the extracellular portions of human respiratory syncytial virus (RSV) F and G glycoproteins. In theory, highly purified FG glycoprotein may be effective as a RSV vaccine. Recombinant FG glycoprotein was expressed using the baculovirus/insect cell system. FG glycoprotein was isolated from cell culture supernatants using S Sepharose ion-exchange chromatography, Cu(2+)-immobilized metal affinity chromatography, preparative reversed-phase high-performance liquid chromatography, denaturation with 6 M guanidine hydrochloride, and protein refolding in Tween 80 detergent. The purified FG glycoprotein was concentrated on a S Sepharose column and exchanged into an appropriate buffer for vaccine formulation. Five batches of FG glycoprotein with protein purity of 92-99% were produced using this purification process. FG glycoprotein produced using reversed-phase chromatography and protein refolding was compared with nondenatured FG glycoprotein using a panel of 14 monoclonal antibodies directed against conformational and linear epitopes on RSV F and G glycoproteins. The results of these studies indicated that refolded FG glycoprotein had the same three-dimensional structure as nondenatured FG glycoprotein.

Large-scale immunoaffinity purification of recombinant soluble human antigen CD4 from Escherichia coli cells.

A large-scale immunoaffinity (IA) purification process was developed for the isolation of recombinant soluble antigen CD4 (sCD4) from Escherichia coli fermentations. The monoclonal antibody used for IA purification of sCD4 recognized a conformation-dependent epitope on the surface of domain 1 of CD4. IA chromatography was used to purify both sCD4-183, consisting of the N-terminal 183 amino acids of human CD4, and sCD4-PE40, a fusion protein consisting of the N-terminal 178 amino acids of CD4 and amino acids 1-3 and 253-613 of Pseudomonas exotoxin A (PE40). sCD4-183 was purified from E. coli cell pellets using cell disruption, protein solubilization, oxidation, Q-Sepharose anion-exchange and IA chromatography steps. sCD4-PE40 was purified from cell pellets using cell disruption, protein solubilization, oxidation, Cu(2+)-immobilized metal-affinity chromatography, anion-exchange and IA chromatography steps. The IA-purified sCD4 analogues demonstrated the correct apparent molecular masses on SDS/PAGE. The immobilized monoclonal antibody appeared to select for correctly folded CD4 protein, since sCD4-183 and sCD4-PE40 purified by the IA method bound human-immunodeficiency-virus glycoprotein gp120 (HIV gp120) in vitro. sCD4-PE40 purified by IA chromatography also inhibited protein synthesis in CV-1 cells expressing HIV gp120/160 at the cell surface. Relatively high recoveries of sCD4-183 and sCD4-PE40 were observed in the IA step of the purification process (71 and 79% recovery respectively). The results demonstrate that immobilized monoclonal antibodies directed against conformational epitopes may be used for rapid purification of gram amounts of correctly folded protein from mixtures of oxidized E. coli proteins.

Recombinant truncated thrombospondin-1 monomer modulates endothelial cell plasminogen activator inhibitor 1 accumulation and proliferation in vitro.

The angiogenic and malignant phenotypes of hamster tumor cells are inversely correlated with the expression of an amino terminally truncated thrombospondin (TSP) subunit. In the present study, we have constructed a truncated TSP subunit from a human fibroblast cDNA library (rt-TSP1) and expressed it in Chinese hamster ovary (CHO) cells. Increased concentrations of plasminogen activator inhibitor-1 (PAI-1) were detected in endothelial cell conditioned medium following treatment with rt-TSP1. This rt-TSP1-induced increase in PAI-1 was neutralized by monoclonal antibodies to both TSP and TGF beta. rt-TSP1 also inhibits the proliferation of endothelial cells and this response is also neutralized by TSP and TGF beta antibodies. Serine and cysteine proteases inhibitors were used to determine if rt-TSP1 activated the latent TGF beta. However, these protease inhibitors did not neutralize the effect of rt-TSP1. The data indicate that the anti-angiogenic properties of TSP may be due to inhibition of the pericellular proteolysis required for endothelial cell migration and endothelial cell proliferation.

A human respiratory syncytial virus (RSV) primate model of enhanced pulmonary pathology induced with a formalin-inactivated RSV vaccine but not a recombinant FG subunit vaccine.

Human respiratory syncytial virus (RSV) is the leading cause of severe bronchiolitis and pneumonia in infants. RSV vaccine development has been stifled for the past 23 years because infants vaccinated with formalin-inactivated (FI) RSV have experienced exacerbated disease upon RSV infection. This exacerbated disease phenomenon is poorly understood, in part because of the lack of a primate model that exhibits a similar exacerbated disease state. Vaccination of African green monkeys with either FI RSV or a genetically engineered subunit vaccine termed FG glycoprotein reduced replication of challenge virus. However, only vaccination with FI RSV induced an enhanced pulmonary pathologic response to RSV infection. Pulmonary inflammatory scores in the FG glycoprotein-vaccinated monkeys were no greater than in monkeys vaccinated with adjuvant alone. This is the first demonstration of RSV vaccine-induced enhanced pathology in a primate and illustrates that a subunit vaccine has the potential of circumventing this exacerbated disease phenomenon.

Topical nonsteroidal agents and corneal wound healing.

The effects on corneal wound healing of two topical nonsteroidal anti-inflammatory agents, flurbiprofen sodium (0.03%) and diclofenac sodium (0.1%), and the topical corticosteroid, prednisolone sodium phosphate (1%), were evaluated in masked, controlled rabbit studies. Healing of epithelial scrape wounds was significantly retarded in all three treatment groups for the first 3 days after wounding. There was no difference in the epithelial healing rate between the two nonsteroidal or corticosteroid treatment groups. Clinical grading of epithelial quality, conjunctival hyperemia, keratitis, stromal edema, and corneal haze were similar in all groups. There was a significant early decrease in the iritis score in the diclofenac treatment group. The strength of 2-mm central penetrating corneal trephination wounds and the collagen content of these wounds were similar in all groups. Both the topical nonsteroidal anti-inflammatory agents and the corticosteroid used in the preparations and dosages investigated in this study decreased early epithelialization of scrape wounds but had no apparent effect on corneal stromal healing. No toxic effects of the various drugs were found.

Histology and immunohistology of Igh-1-restricted herpes simplex keratitis in BALB/c congenic mice.

In order to characterize the local ocular immunologic milieu of Igh-1-restricted herpes simplex keratitis (HSK), we investigated histologic and immunohistologic correlates of disease over a 21-day time course. Clinically observable keratitis began 10 days postinoculation in susceptible C.AL-20 (Igh-1d) and moderately susceptible BALB/c (Igh-1a) mice, whereas HSV-1-resistant C.B-17 (Igh-1b) mice rarely developed disease. Igh-1-restricted histologic differences were observed by day 11 postinoculation; C.AL-20 and BALB/c mice showed augmented recruitment of neutrophils and mononuclear cells in conjunctival, limbal, and corneal tissues compared to C.B-17 mice. On immunohistologic study, Lyt-1 to Lyt-2 cell ratios by day 11 postinoculation were 7:1, 2:1, and 1:8 in corneas from C.AL-20, BALB/c, and C.B-17 mice, respectively. Macrophages and neutrophils were absent in corneas from C.B-17 mice at this time, but could be found in large numbers in the corneas of susceptible mouse strains through day 21. These data demonstrate a strong relationship between Igh-1 phenotype and inflammatory cell recruitment in response to corneal infection with HSV-1, and support a role for T cell subpopulations in mediating Igh-1-restricted HSK.

Cyclic nucleotide modulation of herpes simplex virus latency and reactivation.

Clinical observations and experimental studies suggested that the relative proportions of ganglionic neuronal intracellular cyclic adenosine monophosphate (c-AMP) and cyclic guanosine monophosphate (c-GMP) concentrations may influence the state or activity of herpes simplex viral DNA in its relationship with the host cell DNA. We studied the effects of putative modulators of intracellular cyclic nucleotide levels on herpes simplex virus (HSV) reactivation from latency in murine trigeminal ganglion cells. We also investigated the effects of these same mediators on the c-GMP and/or c-AMP concentrations in HSV-latently infected trigeminal ganglion cells and in acyclovir-suppressed, HSV-infected neuroblastoma cells. Cholera toxin and theophylline increased c-AMP levels (2-fold and 5-fold at 1 min and 30 sec, respectively for cholera toxin and 2-fold and 1.5-fold at 1 min and 30 sec for theophylline) and enhanced the rapidity of HSV reactivation from latency (P less than 0.005). Exogenous dibutyryl c-AMP also stimulated viral reactivation (P less than 0.005). Carbamylcholine increased c-GMP levels (7-fold and 6-fold at 15 sec and 30 sec, respectively), produced no significant change in c-AMP levels, and delayed HSV reactivation from latency (P less than 0.005). None of these mediators had a demonstrable effect on HSV replication.

Rat bioassays for methionine availability in 16 food sources.

Methionine availabilities of 16 test proteins were assessed by comparing ten day rat growth response to the test diets and reference (casein) diets. In a preliminary study, various concentrations of methionine and cystine were fed to determine methionine requirements and effect of excess cystine. Results indicated a methionine requirement of about 550 mg per 100 g diet. Cystine had a sparing affect of 50-55%, i.e., about 300 mg could be used to meet methionine requirements. Further additions of cystine (up to 2.6 times methionine) did not affect rat growth. Methionine availabilities were excellent (88-100%) for 15 of the 16 test foods; only pinto beans (58%) were low, but prior evidence indicates that the poor growth response was due to some factor other than availability.

Bioavailability of tryptophan in selected foods by rat growth assay.

Tryptophan bioavailabilities were estimated in 16 protein sources using 10 day rat growth assays with casein as the reference protein. Growth responses of rats fed test food diets were compared to growth responses of rats fed basal diets with graded levels of tryptophan ranging from 50 to 100 mg of tryptophan/100 g diet. Estimates of tryptophan availabilities were 85-100% for all products except whole wheat cereal (73%) and pinto beans (59%). Results of a previous study on lysine availability indicated that poor response to pinto beans was due either to poor digestibility or to the presence of some unidentified growth inhibitor.

The use of Escherichia coli mutants to measure the bioavailability of essential amino acids in foods.

The beta-galactosidase-based assay for lysine developed by Tuffnell & Payne was used to measure the bioavailabilities of cyst(e)ine, methionine, threonine and tryptophan in pronase digests of 17 foods. The digests were assayed by estimating the beta-galactosidase synthesis responses of five Escherichia coli mutants, each requiring one of the respective amino acids for protein synthesis. Deletion mutants were used whenever possible in order to ensure strain stability. Single digests of each food were assayed with 3 or 4 separate cultures of each mutant and the results were compared with those from the corresponding chemical assay. Omitting the anomalously low values for one food, the rank correlation coefficients of the bio- and chemo-assay values were 0.61 (cysteine), 0.91 (lysine), 0.95 (methionine), 0.64 (threonine) and 0.85 (tryptophan). Mean (+/- S.D.) relative amino acid bioavailabilities (casein = 100%) for the 17 foods were: cysteine, 53 +/- 23; lysine, 90 +/- 10; methionine, 95 +/- 18; threonine, 89 +/- 13; and tryptophan, 89 +/- 25. The cysteine mutant appeared to give unusually low bioavailability values except for the milk products. These amino acid mutants afford a rapid method for assaying the bioavailabilities of at least four of the five amino acids studied.

Immunomodulation of experimental murine herpes simplex keratitis: I. UV-HSV protection.

A/J mice were immunized subcutaneously with ultraviolet light (UV) inactivated herpes simplex virus type-1, MP strain (HSV-MP). Control A/J mice were immunized subcutaneously either with media (unimmunized controls) or with live HSV-MP (immunized controls). Immunized and control mice were challenged ocularly with either MP or mP strain HSV-1 after corneal scarification and were followed for 3 weeks post corneal challenge. The mice were observed during this time period for signs of herpes simplex keratitis (HSK), lid lesions and encephalitis. At the time of sacrifice, the ipsilateral trigeminal ganglia were removed and assayed for latent HSV-1 using cocultivation on Vero cell monolayers. The results of these studies demonstrated that immunization with UV inactivated HSV (UV-HSV) gave the same protection against keratitis and encephalitis as immunization with live virus. Furthermore, the cocultivation assays indicated that immunization with either live HSV-1 or UV inactivated HSV-1 protected against the establishment of latency.

Immunomodulation of experimental murine herpes simplex keratitis: II. Glycoprotein D protection.

Subcutaneous immunization with purified HSV-1 glycoprotein D (gD) protects susceptible A/J mice against keratitis and encephalitis following corneal HSV-1 challenge. Mice were immunized with gD, in complete Freund's adjuvant, 3.0 micrograms/mouse followed by two booster doses of 1.5 micrograms/mouse at weeks 2 and 4. Control groups of A/J mice were injected with either complete Freund's adjuvant (unimmunized controls) or live HSV-1 MP strain (immunized controls). All mice were challenged ocularly at week 5 with HSV-1, F strain (6.5 x 10(3) PFU) after corneal scarification. None of the 16 animals immunized with gD developed stromal keratitis; only 3 out of 12 animals immunized with live HSV-1 developed a stromal keratitis; 13 out of 16 CFA primed unimmunized mice developed severe stromal keratitis within 14 days post corneal challenge, and 3 out of 16 control CFA primed animals died within 16 days post corneal challenge. At the time of sacrifice (3 weeks post corneal challenge), the ipsilateral trigeminal ganglia were removed and assayed for latent HSV-1 using cocultivation on Vero cell monolayers. The results of these experiments indicate that immunization with gD produces protection against latent ganglionic infection in 56% of the immunized animals, and provides protection against keratitis and death following HSV corneal challenge.

Effect of Intralipid, amino acids, container, temperature, and duration of storage on vitamin stability in total parenteral nutrition admixtures.

This study was designed to determine the stability of certain vitamins added to total parenteral nutrition (TPN) admixtures with or without Intralipid iv fat emulsion and with each of four amino acid solutions stored in either glass bottles or plastic bags at either ambient room (25 degrees C) or refrigerator (5 degrees C) temperature for a 48-hr period. Riboflavin and folacin were not affected by the experimental conditions. The presence of Intralipid resulted in higher levels of vitamin E due to Intralipid's inherent vitamin E content; no other experimental conditions affected vitamin E. Thiamin levels decreased in admixtures containing the amino acid solution C and stored at 25 degrees C. Vitamin A levels were lower in admixtures stored in plastic but were maintained in admixtures containing Intralipid and stored in glass bottles at either temperature. Vitamin C levels were maintained in admixtures stored at 5 degrees C for all experimental conditions. The greatest vitamin C losses occurred in admixtures containing amino acid solutions C or D stored in plastic bags, or containing D stored in glass bottles at 25 degrees C.

Effect of phototherapy light, sodium bisulfite, and pH on vitamin stability in total parenteral nutrition admixtures.

The three sections of this study extend previous research into losses of vitamins A, C, E, thiamin, riboflavin, and folic acid from total parenteral nutrition (TPN) admixtures. First, phototherapy light on TPN admixtures containing one of four amino acid solutions was studied. Experimental conditions included presence or absence of Intralipid iv fat emulsion, plastic bag or glass bottle storage container, and storage time of up to 48 hrs. The second phase studied stability of the same vitamins (except vitamin E) for 48 hrs in admixtures containing the amino acid solution which has no bisulfite, in glass bottles; with or without Intralipid; and with added sodium bisulfite (final concentrations of 0, 1, 2, 3, 4, 5 and 10 mEq/liter). Third, vitamin C and thiamin levels were measured in admixtures containing the amino acid solution with no bisulfite, without Intralipid, stored in glass bottles with various bisulfite concentrations (0, 1, 2, or 3 mEq/liter) and three pH levels (5.5, 6.5, and 6.75 pH). Exposure of TPN admixtures to phototherapy light caused losses of vitamins A, C, and riboflavin. Intralipid inclusion significantly reduced losses of vitamin A and riboflavin, but did not appear to affect vitamin C levels. The smallest vitamin C losses were noted in admixtures containing amino acid solutions A or B. Phototherapy light did not affect thiamin levels. Bisulfite had no affect on vitamin C, riboflavin, or folic acid levels. Vitamin A levels were maintained with bisulfite concentrations less than 3 mEq/liter. At 3 mEq/liter bisulfite, admixtures with Intralipid showed 50% loss of vitamin A.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunogenetic influence of Igh-1 phenotype on experimental herpes simplex virus type-1 corneal infection.

Patterns of herpes simplex virus type-1 (HSV-1) infection were studied in BALB/c congenic, Igh-1 disparate murine strains to establish the influence of Igh-1 phenotype on the development of keratopathy, trigeminal ganglionic latency and keratocyte permissivity. Eighty-two percent of C.AL-20 (Igh-1d) mice, 40% of BALB/cByJ (Igh-1a) mice and 12% of the C.B-17 (Igh-1b) mice developed herpes simplex keratitis (HSK) following corneal challenge with 2.5 X 10(4) PFU HSV-1 strain KOS. While disease frequency was directly proportional to HSV-1 challenge dose, relative resistance and susceptibility patterns in the congenic mice were constant and highly significant. F1 progeny from C.AL-20 X C.B-17 matings demonstrated the HSK pattern of the C.B-17 parent suggesting that Igh-1 linked resistance to HSK is dominantly inherited. Equivalent trigeminal ganglionic latency was established following ocular HSV-1 inoculation in the three congenic Igh-1 disparate murine strains. Cultured keratocytes from the three Igh-1 disparate murine strains demonstrated equivalent in vitro permissivity to HSV-1 replication. These data illustrate a strong correlation between Igh-1 phenotype and the development of a HSK in congenic mice. The susceptibility/resistance to HSK in these mice is unrelated to trigeminal ganglionic latency or keratocyte permissivity.

Detection of ocular mucus in normal human conjunctiva and conjunctiva from patients with cicatricial pemphigoid using lectin probes and histochemical techniques.

Conjunctival biopsies from patients with cicatricial pemphigoid affecting the conjunctiva and patients undergoing cataract surgery (normal conjunctiva) were snap-frozen, cryostat sectioned and incubated with fluorescein-conjugated lectins; peanut agglutinin (PNA), Helix pomatia agglutinin (HPA), soybean agglutinin (SBA), wheat germ agglutinin (WGA) and succinylated wheat germ agglutinin (S-WGA). Controls consisted of preincubating the lectins with the appropriate blocking sugars before applying the lectins to the sections. PNA and HPA stained the mucus granules contained in the conjunctival goblet cells but did not stain mucus or glycocalyx at the ocular surface distal to the goblet cells. Native WGA and S-WGA had high affinities for conjunctival goblet cells and the apical epithelial cell layers. Native WGA stained mucus and glycocalyx at the ocular surface. This staining of the ocular surface by WGA was confirmed at the transmission electron microscopic level using WGA conjugated to ferritin. Cicatricial pemphigoid patients in this study had reduced numbers of goblet cells; however, those goblet cells which were observed in cicatricial pemphigoid conjunctiva stained positively with HPA, PNA, WGA, and SWGA as did goblet cells in normal conjunctiva.